This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In our COBRE project we will study the ionotropic cannabinoid receptor, TRPV1, which is a critical determinant of calcium entry and mast cell activation in response to both cannabinoids and physico-chemical signals. Our data show that polybasic secretagogues, which drive chymase release from mast cells, act via TRPV1 using a Gai/o-coupled pathway. Expression of TRPV1 is necessary and sufficient to confer secretagogue-mediated calcium entry on cells that are natively unresponsive to secretagogue. Secretagogue application, and exposure to an acidified environment, have recently been implicated in a specialized function of mast cells: the release of proteinases that activate the Renin-Angiotensin System (RAS) in tissues such as the heart and lung. TRPV1-mediated calcium entry drives chymase release from mast cells in response to secretagogues. These data implicate TRPV1 in the promotion of pro-hypertrophic mediator release from mast cells. Moreover, our data suggest that TRPV1 integrates both pro- and anti-hypertrophic signals. The ANP receptor guanylyl cyclase-coupled A (GC-A, NPR1) physically associates with TRPV1 and we have shown that GC-A mediates cGMP/PKG-mediated suppression of TRPV1 activity. These data implicate TRPV1 in responses to stimuli that both promote, and oppose, the secretion of pro-hypertrophic mediators from mast cells. Although mast cells are best studied and understood in dermal and mucosal immune systems, they are present in the myocardium where they are thought to play a role in inflammation. The central premise of this proposal is that mast cells are sources of cytokines, matrix-active enzymes and pro-inflammatory factors that help maintain homeostasis in the healthy heart, but contribute to inappropriate tissue re-modeling in the failing heart. A common mechanism of all mast cell activation is calcium influx through either TRPV1, in response to non-immunological stimulation, or CRACM1, in response to immunological stimulation. It is our overarching hypothesis that it is TRPV1 that is responsible for calcium influx in mast cells in the failing heart. We therefore plan to investigate the degree to which mast-cell TRPV1 and CRACM1 contribute to tissue modeling and pathology in the failing heart with our three specific aims. Aim 1. Determine the role of mast cells, TRPV1, and CRACM1 in the failing heart. We will evaluate mast cell signaling in a model of heart failure induced by aortic constriction. Assessment of basic cardiac physiology will be performed, using histological, functional, and transcriptional assays, of hearts from TRPV1, CRACM1 and mast cell deficient (KitW-sh/W-sh) mice undergoing these treatments. Aim 2. Examine the effects of restoring the mast cell-related contribution to the failing heart by reconstitution of mast cell-deficient mice. To control for the effects of TRPV1 and CRACM1 knockout in the majority of non-mast cell tissues, we will reconstitute lethally irradiated mice with bone marrow from TRPV1 and CRACM1 knockout mice. After reconstitution, heart failure will be modeled as in Aim 1. Aim 3. Test whether pharmacologic modification of TRPV1, CRACM1 and mast cell activation can ameliorate pressure overload-induced cardiac failure. Modulation of mast cell activation may influence remodeling in heart failure. There is an extensive pharmacopeia that regulates TRPV1 and we plan to test the effects of these drugs on anatomic, functional, and histological progression in a model of pressure-induced cardiac failure. We expect to determine whether current small molecule TRPV1 and ICRAC/CRACM1 antagonists could have therapeutic utility in heart failure.